Project Summary/Abstract Glioblastomas remain one of the most deadly cancers with no breakthroughs in therapy for the past 20 years. Xandra Breakefield has made critical discoveries demonstrating that these tumor cells release extracellular vesicles containing informative nucleic acids and proteins that convert normal brain cells to tumor supportive cells. Working with a team of seasoned investigators she has continued to make breakthrough advances in the use of these vesicles as biomarkers, in elucidating the means by which they subjugate microglia and other cells in their microenvirons, and in exploring how they may be channeled for therapeutic purposes. This team consisting of Drs. Breakefield, Joseph El Khoury/Suzanne Hickman (microglia experts), Thorsten Mempel (T lymphocyte expert), Marike Broekman (neurosurgeon) and Casey Maguire (vector expert) will advance these insights in three interrelated areas: biomarkers, cell-to-cell communication and therapy. Studies are designed to increase sensitivity and reveal clinical correlates of RNA and protein in extracellular vesicle biomarkers from serum/plasma with goals of early detection, informing therapeutic decisions and longitudinal evaluation. They will explore how tumor extracellular vesicles participate in changing the phenotype of microglia, macrophages and astrocytes in the tumor microenvirons, such that they become a ?life support? system for the tumor in defiance of therapy. These insights will be forged into new therapeutic concepts with a focus on engaging the innate and adaptive immune systems to arm the brain against the tumor. This will include increasing cross presentation of extracellular vesicle-derived tumor antigens via microglia to infiltrating T lymphocytes using co- stimulatory molecules. Microglia associated with the tumor will be endowed with increased capacity to release anti-microbial peptides, which are also anti-tumorigenic, to reawaken their sense of the presence of the tumor and to down-regulate program death-ligands that exhaust cytotoxic T lymphocytes. Tumor-associated cells, including reactive astrocytes, microglia and macrophages will be manipulated using systemically administered adeno-associated virus and other vectors (which are clinically compatible) carrying transgene cassettes under promoters that are strongly up-regulated in cells near the tumor, but not in the same cell types in other parts of the brain. Vesicles produced by these cells will also be used to deliver therapeutic cargo to tumors cells to provide sustained therapeutic impact. These studies acknowledge the cytoplasmic continuum of cancer among all the cell types that make up the tumor mass, and strike at this supportive microenvironment which sustains the tumor. Therapeutic strategies are designed to be combinatorial with standard-of-care without increasing morbidity for this devastating disease that has defied current therapeutic approaches.